The RT-LAMP-LFD Detection Method,Epidemiological Investigation And Some Biological Characteristics Of Senecavirus A

Senecavirus A（SVA）is a new infectious RNA virus,also a pathogen causing vesicular symptoms in pigs and increased acute mortality of piglets.In recent years,the virus has caused economic losses to farms in at least seven countries and at least 14 provinces or autonomous regions in China.In order to establish a molecular detection method suitable for clinical diagnosis and rapid detection of SVA,a RT-LAMP-LFD method was established.The established RT-LAMP-LFD detection method can directly determine the results on the LFD visualization strip after 50 minutes of reaction at 61℃.The detection limit of this method was 4.5x10^-8ng/μL RNA（about 11 copies）,and its sensitivity was 10 times higher than that of RT-PCR.When this method was applied to clinical samples,the detection rate of SVA was comparable to that of RT-PCR,but it has the advantages of rapid detection and high efficiency of nucleic acid amplification,so it can be used to diagnose suspected SVA infection.In order to reveal the prevalence of SVA in pigs with five different clinical manifestations and the prevalence of SVA in some regions of China,916 clinical samples were collected from pigs with different clinical manifestations in 14 different geographical regions of China.Then,RT-PCR was used to detect these samples.The results showed that there were cases of SVA infection in both groups with and without vesicular signs,and their positive rates of SVA were 53.33%（64/120）and 9.30%（74/796）,respectively.The positive rate of SVA was higher in the group without vesicles but with diarrhea（21.55%,39/181）and lowest in asymptomatic pigs（0.64%）.SVA infection was also found in pigs with reproductive and respiratory symptoms,but the data showed no statistical significance（P>0.05）.This study showed that SVA was prevalent in pig herds with five different clinical manifestations sperately,which suggests that although the main clinical manifestations of SVA infection are vesicular signs,SVA could also be detected in the samples with the above clinical manifestations.In addition,the study also found that there were different levels of SVA infection rate（0%～100%）in 14 different geographical regions of China,while the average positive rate of SVA in the survey areas was 15.07%（138/916）.This finding provides a reference for SVA prevention and control in some areas of China.In this study,VP1 gene fragments of 7 SVA strains and full-length sequences of 3 SVA strains were amplified.The nucleotide similarity of the VP1 fragments between the seven SVA strains and the early SVA strain 88-23626（EU271759）was 86.4%～87.7%,and the amino acid similarity was 65.7%～69.0%;while the nucleotide similarity with the first Chinese SVA strain CH-01-2015（KT321458）was 95.3%～95.9%,and the amino acid similarity was 88.1%～90.5%.The genetic evolution analysis based on VP1 showed that the seven strains belonged to three different sub clades.Furthermore,the genetic evolution analysis based on the complete genome showed that the three SVA strains（1/2018/HZ/China,1/2018/BH/China and GDHY/2018）were grouped into three different sub clades respectively,and all of them have relatively close genetic relationship with some SVA strains reported in recent years in China.This study revealed the genetic diversity of SVA strains in South China,and it would provide a reference for a more comprehensive epidemiological survey of SVA.In this study,the SVA strain HN16 isolated in 2016 was cultured in vitro.It was found that HN16 strain had fully adapted to the subculture of PK-15 cell lines and BHK-21 cell lines.The time of cytopathy was shortened,which made it possible that more than 90%of cytopathy could be observed in 30 hpi.Through the plaque purification experiment,HN16strain was successfully purified.The homology of the complete genome between F110 and its original sequence was 99.7%,which indicated that the gene sequence of the strain was relatively stable in the process of continuous passage.In this study,a purified SVA strain HN16,which with high virus titer and stability after culture and passage,was obtained.This strain is expected to be a candidate for SVA attenuated or inactivated vaccine.